| ObjectiveLXR-623(WAY-252623),a liver X receptor agonist,reduces atherosclerotic plaque progression and remarkably inhibits the proliferation of glioblastoma cells,owing to its brain-penetrant ability.However,the role of LXR-623 against the proliferation of other cancer cells and the underlying mechanism remain unknown.Long non-coding RNAs(lncRNAs)serve as novel and crucial regulators that participate in cancer tumorigenesis and diverse biological processes.Here,we report a previously uncharacterized mechanism underlying lncRNA-mediated exocytosis of LXR-623 via the phosphatase and tensin homolog(PTEN)/protein kinase B(AKT)/p53 axis to suppress the proliferation of cancer cells in vitro.We found that LXR-623 significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at S phase in breast cancer cells in a concentration-and time-dependent manner.Experiments using a xenograft mouse model revealed the inhibitory effects of LXR-623on tumor growth.We used lncRNA microarray to investigate the potential genes regulated by LXR-623.As a result,LINC01125 was found to be significantly upregulated in the cells treated with LXR-623.Gain-and loss-of-function assays were conducted to investigate the anti-proliferation role of LINC01125.LINC01125 knockdown resulted in the inhibition of the cytotoxic effect of LXR-623;in contrast,LINC01125 overexpression significantlyenhancedtheeffectofLXR-623.LXR-623and LINC01125-mediated anti-growth regulation is,at least in part,associated with the participation of the PTEN/AKT/mouse double minute 2 homolog(MDM2)/p53 pathway.In addition,SF1670,a specific PTEN inhibitor with prolonged intracellular retention,may strongly block the anti-proliferation effect induced by LXR-623 and LINC01125 overexpression.Chromatin immunoprecipitation(ChIP)assay results suggest that p53 binds to the promoter of LINC01125 to strengthen the expression of the PTEN/AKT pathway.Taken together,our findings suggest that LXR-623 possesses significant antitumor activity in breast cancer cells that is partly mediated through the upregulation in LINC01125 expression and enhancement in apoptosis via the PTEN/AKT/MDM2/p53 pathway.MethodThis study is divided into two parts.1.LXR-623 inhibits proliferation and promotes apoptosis of breast cancer in vivo and in vitro(1)Effects of LXR-623 on proliferation and apoptosis of breast cancer cells1)CCK8 detection was used to detect the proliferation and apoptosis of LXR-623 for different concentrations(1,2.5,5?mol/l)and different time(24,48,72h)on breast cancer cell lines:MDA-MB-231 BT-549,MCF7,MDA-MB-453,compared to normal mammary epithelial cells MCF10A.2)Flow cytometry,cycle detection and colony formation experiments were used to detect the apoptosis of LXR-623(5?mol/l)for different treatment time(24,48,72 h)for breast cancer cell lines:MDA-MB-231,BT-549.3)Western blot was used to detect the expression of apoptosis-related protein:Cleaved Caspase3,Bax,BCL2 and cell cycle related proteins:expression of cyclin E1,CDK2,and cyclin A2 in the concentration on LXR-623(5?mol/l)in different time(24,48,72 h)in breast cancer cell lines:MDA-MB-231,BT-549,respectively.(2)Effects of LXR-623 on the growth of xenograft tumors1)LXR-623 was orally administered to nude mice inoculated with MDA-MB-231 cells,and the effect of LXR-623 on the growth of transplanted tumors was observed.2.Therapeutic Target and downstream Pathway Mechanism of LXR-623inhibiting the Proliferation of Breast Cancer cells.(1)lncRNA chip was used to explore the pharmacodynamic target of LXR-623 and the functional phenotype of lncRNA molecule in breast cancer.The main results were as follows:1)lncRNA microarray was used to explore the differentially expressed lncRNA molecules in MDA-MB-231 cells treated with LXR-623.2)the selected lncRNA molecule,LINC01125,was detected by qPCR for the expression of LINC01125 in breast cancer patients,the expression of LINC01125 in breast cancer cell lines and the localization of LINC01125in breast cancer cells detected by FISH.3)the proliferation and apoptosis of Vector group,overexpression LINC01125 group,overexpression LINC01125+LXR-623 group and si-NC group,si-LINC01125 group and si-LINC01125++LXR-623 group were detected by CCK8 and flow cytometry,respectively.4)Western blot was used to detect apoptosis-related proteins:Cleaved caspase3,Bax,BCL2 and cell cycle related proteins:cyclin E1,CDK2,cyclin A2 in Vector group,overexpression LINC01125 group,overexpressionLINC01125+LXR-623groupandsi-NCgroup,si-LINC01125 group and si-LINC01125++LXR-623 group.(2)downstream pathway mechanism of LXR-623.1)Western blot was used to detect the expression of PTEN,AKT,p-AKT,MDM2,p-MDM2 and p53 in MDA-MB-231 and BT-549 cell lines with or without the treatment for LXR-623.2)After adding PTEN specific inhibitor SF1670,CCK8 and flow cytometry were detected the proliferation and apoptosis of DMSO group,LXR-623+SF1670 group and LXR-623 group.3)Western blot was used to detect the expression of apoptosis-related proteins:Cleaved caspase3,Bax and BCL2 in SF1670 group,LXR-623+SF1670 group and LXR-623 group.(3)the regulatory relationship between LINC01125 and PTEN/AKT/p53pathway.1)Western blot was used to detect the expression of PTEN,AKT,p-AKT,MDM2,p-MDM2 and p53 in non-transfaction group,Vector group,overexpression LINC01125 group and non-transfaction group,si-NC group and si-LINC01125 group.2)After adding PTEN specific inhibitor SF1670,CCK8 and flow cytometry were used to detect the proliferation and apoptosis of Vector+SF1670 group,overexpression LINC01125+SF1670 group and overexpression LINC01125 group.3)Western blot was used to detect the expression of PTEN,AKT,p-AKT,MDM2,p-MDM2 and p53 in Vector+SF1670 group,overexpression LINC01125+SF1670 group and overexpression LINC01125 group.4)Bioinformatics predicts the direct regulatory relationship between LINC01125 and p53.Result1.Inhibitory effect of LXR-623 on proliferation and apoptosis of breast cancer in vivo and in vitro.(1)the effect of LXR-623 on the proliferation and apoptosis of breast cancer cells.1)To evaluate the effect of LXR-623 on BC cells and a normal epithelial breast cell line MCF-10A,we measured the cell viability using the cell counting kit 8(CCK8)assay after the treatment of cells with LXR-623 at different concentrations(1,2.5,and 5?mol/l)for 24,48,and 72 h.As a result,we found that LXR-623 significantly suppressed the viability of MCF?7,MDA?MB?231,BT549,and MDA-MB-453 cells in a dose?and time?dependent manner(P<0.05).2)Flow cytometry,cell cycle assay and clone formation assay were used to detect the number of apoptotic cells after exposure to LXR-623 at 5?mol/l for 24,48,and 72 h.In comparison with the cells treated with dimethyl sulfoxide(DMSO;control group),MDA-MB-231 and BT549 cells treated with LXR-623 for 72 h showed almost 60%increase in the rate of apoptosis.In addition,we investigated the effect of LXR-623 at 5M concentration for 24,48,and 72 h on cell cycle progression by flow cytometry and observed cell cycle arrest at the S phase in MDA-MB-231and BT549 cells as compared to the control groupThe colony formation assay revealed the significant inhibition in the colony-forming ability of MDA-MB-231 and BT549 cells after treatment with the tested drug at a concentration of 1,2.5,and 5?mol/l.(P<0.05).3)We tested the expression of apoptosis-related proteins by western blot analysis and found that the expression of activated(cleaved)caspase-3 and BAX was upregulated and that of B-cell lymphoma 2(BCL2)was downregulated in a time?dependent manner.As LXR-623 may induce cell cycle arrest,we tested the protein levels of cyclin E1,cyclin-dependent kinase 2(CDK2),and cyclin A2 with western blot analysis and found that the expressions of cyclin E1 and CDK2 were upregulated and cyclin A2expression was downregulated(2)Antitumor effects of LXR-623 in vivo.To validate the antitumor effect of LXR-623 in vivo,female nude mice were subcutaneously injected with MDA-MB-231 cells to induce tumor formation.The mice were randomly divided into two groups once the tumor volumes reached about 100 mm~3.During the treatment period of 10days,tumor weight and volume significantly decreased for mice treated with LXR-623 as compared to those from the control group.2.Therapeutic target and downstream pathway mechanism of LXR-623(1)lncRNA microarray was used to explore the target of LXR-623 and the functional phenotype of lncRNA molecule in breast cancer.1)To identify the candidate lncRNAs involved in LXR-623-mediated tumor suppression,we used the lncRNA microarray to evaluate the differentially expressed genes regulated by LXR-623 at 5?mol/l concentration for 48 h in MDA-MB-231 cells.lncRNA microarray results revealed significant changes in the expressions of 943 human lncRNAs after treatment with LXR-623.Of these human lncRNAs,433 were upregulated and 510 were downregulated2)qRT-PCR results confirmed the increase in the expression of LINC01125 mediated by LXR-623 in the xenograft tumors as well as MDA-MB-231and BT549 cells in a dose-dependent manner.LINC01125 expression level was downregulated in tumors as compared with the corresponding normal tissues.We used The Cancer Genome Atlas(TCGA)database to analyze the expression of LINC01125 and found that LINC01125 expression was downregulated in BC tissues.qRT-PCR was used to confirm the downregulation of LINC01125 expression in MDA-MB-231,BT549,MCF-7,andMDA-MB-453cellsthaninMCF-10A cells.Immunofluorescence assay consistently showed that LINC01125 was more enriched in the cytoplasm of MDA-MB-231 and BT549 cells.Therefore,LINC01125 may play an important role as a suppressor gene in BC.3)To assess the potential functional role of LINC01125 in LXR-623-induced tumor suppression,we examined the impact of LINC01125 overexpression and knockdown in BC cell lines and found that the knockdown of LINC01125 with a small interfering RNA(siRNA)led to an increase in the proliferation of tumors;however,the treatment of these transfected cells with LXR-623 partially blocked the inhibitory effects of LXR-623,as revealed by the CCK8 and apoptosis assays.In contrast,cell viability significantly decreased for those cells overexpressing LINC01125 as compared with the negative control group.Upon treating cells with LXR-623,the apoptosis rate was dramatically increased.Next,the knockdown of LINC01125 slightly reduced the number of cells in the S phase,but LINC01125 overexpression increased the number of cells in the S phase and strengthened the effect of LXR-623.4)Western blotting results showed that LXR-623 induced the expression level of cleaved caspase-3 and BAX and suppressed the expression of BCL2.This effect may be strengthened through the overexpression of LINC01125 or partly blocked through LINC01125 knockdown.Western blot analysis also showed that LXR-623-induced upregulation in cyclin E1and CDK2 expression and downregulation in cyclin A2 expression could be weakened through the knockdown of LINC01125 or magnified through the upregulation in LINC01125 expression.(2)downstream pathway mechanism of LXR-623.1)To determine the mechanism underlying the anti-proliferation effect of LXR-623 in vitro,we performed western blot analysis.The results showed that the cells treated with LXR-623 at 5M concentration for 24,48,and72 h showed an increase in the expression of PTEN and p53 and reduced the expression of p-Akt and p-MDM2 in a time?dependent manner.2)To examine the regulation of PTEN pathways mediated by LXR-623,MDA-MB-231 and BT549 cells were treated with a PTEN specific inhibitor(SF1670)for 48 h.Then,SF1670 treatment could partly reverse the LXR-623-induced anti-proliferation effect,as revealed from the results of the CCK8 assay and apoptosis experiments.3)We found an increase in the expression of cleaved caspase-3 and BAX and a decrease in BCL-2 expression upon treatment with LXR-623.These effects of LXR-623 could be partly inhibited through SF1670(3)The regulatory relationship between LINC01125 and PTEN/AKT/p53pathway.1)The knockdown of LINC01125 reduced the expression of PTEN and p53 and increased the expression of p-Akt and p-MDM2,while LINC01125 overexpression enhanced the expression of PTEN and p53 and lowered the expression of p-Akt and p-MDM2.2)The LINC01125-mediated anti-proliferation effect was also reversed by SF1670 by CCK8 assay and apoptosis experiments.3)The BC cells treated with SF1670 showed an increase in the expressions of p-AKT and p-MDM2 and a decrease in the expressions of PTEN and p53 upon LINC01125 overexpression,suggestive of a link between LINC01125 and PTEN pathways.4)To investigate the mechanism underlying the regulatory effect of LINC01125 on PTEN pathway,we examined the impact of p53overexpression and knockdown on BC cell lines.Firstly,qRT-PCR results demonstrated that the expression of p53 was significantly induced or reduced in both MDA-MB-231 and BT549 cell lines after its overexpression or knockdown,respectively.CCK8 assay was used to evaluate the viability of the co-transfected MDA-MB-231 and BT549 cells.Treatment with LXR-623 had no effect on the co-transfection of LINC01125 overexpression vector and si-p53 as well as si-LINC01125 and p53 overexpression vector.Furthermore,ChIP assay was performed to evaluate the binding of p53 to the promoter region of LINC01125,with an IgG-precipitated sample used as a negative control.We observed an increase in the binding between p53 and the LINC01125 promoter in the two cell lines.These data suggest that p53 showed significant binding to the promoters of LINC01125.ConclusionTaken together,our findings suggest that LXR-623 possesses significant antitumor activity in breast cancer cells that is partly mediated through the upregulation in LINC01125 expression and enhancement in apoptosis via the PTEN/AKT/MDM2/p53 pathway.Therefore,LXR-623 may become a new target for adjuvant treatment of breast cancer,and it has been proved that lncRNA can play a key role in the occurrence and development of cancer and even a variety of biological reactions. |
PDF Full Text Request